Part:BBa_K5182019
Part of optogenetic modulation of gene therapy system
SJTU-BioX-Shanghai 2024
Composite part BBa_K5182019 is mainly composed of BBa_K3281012, BBa_K5182005, BBa_K5182010, BBa_K5182009 and BBa_K5182003
Background
Our optogenetic based therapeutic modulation system consists of three composite parts BBa K5182017, BBa K5182018, BBa K5182019 together
In recent years, there has been preliminary progress in the application of light-based gene expression regulation systems in mammalian cells. These systems utilize photosensitive receptors and light-regulated proteins to control the timing and intensity of gene expression, providing a new method for precisely manipulating the expression of target genes[1]. Particularly in gene therapy, light-regulated systems allow treatments to be activated only when needed, thereby reducing the risk of side effects and increasing the controllability of the therapy[2].
We innovatively introduced the UV receptor system from plants into the gene therapy strategy, using UV-B light as an external regulatory factor to induce the expression of the gene. The UVR8 receptor, which is widely present in plants, is sensitive to UV-B light, and its activation can trigger a series of downstream signaling processes[3]. By translating this mechanism into mammalian cells, we can control the expression of the gene using UV-B light.
Fig1. UVR8-COP1 module UVB signaling response mechanism
In plant, COP1 is an E3 ubiquitin ligase that targets HY5 for degradation[4]. UVR8 is a photoreceptive protein that forms a homodimer outside the cell nucleus in the absence of UV-B signals. When UV-B signals are present, UVR8 monomerizes and enters the nucleus, where it interacts with COP1, playing a role in plant photomorphogenesis. RUP2 is a core protein in plants that regulates the response to UV-B radiation, serving as a negative regulator in the UV-B signaling pathway[5]. Therefore, our project primarily utilizes the interaction between UVR8, COP1 and RUP2 in response to UV-B signals to initiate the expression of the target gene.
Fig2. negative feedback regulation mechanism of RUP2 in the UVR8-COP1 optogenetic system
Fig3. Schematic diagram of the UV-responsive XPC gene switch. The position of the XPC gene can be replaced with other genes of interest to the creator
Design
Gal4 is a transcriptional activator derived from yeast, typically composed of two domains: the DNA-binding domain (BD) and the transcriptional activation domain (AD)[6]. It can recognize and bind to the upstream activation sequence (UAS), thereby activating the transcription of downstream genes. Our project utilizes the BD domain of Gal4 and the 5UAS sequence to facilitate protein localization through the binding of Gal4 to 5UAS. VP64 is a potent transcriptional activator that, when bound to the promoter, can activate gene transcription. The P2A peptide is a self-cleaving peptide that enables the independent translation of two genes located before and after the P2A sequence[7].
Fig4. the binding of GAL4 with 5×UAS
Fig5. the strong transcriptional activator VP64. VP64 is a potent transcriptional activator that binds to RNA polymerase II (Pol II) and activates gene transcription by increasing the stability of the transcription initiation complex and facilitating transcription initiation.The structure of VP64 allows it to enhance the transcriptional activity of genes by recruiting additional transcription factors and cofactors through interactions with transcription factors.
We have fused UVR8 with VP64 and COP1 with BD domain of Gal4, added a nuclear localization sequence (NLS) behind COP1 to stabilize its location in the cell nucleus, which is more conducive to initiating gene expression. The target XPC gene is connected behind the 5UAS sequence and the CMV promoter, and the RUP2 is connected behind the XPC gene through the P2A sequence. Gal4 tightly binds to 5UAS within the nucleus, and when UV-B signal is present, UVR8 monomerizes and enters the nucleus to interact with COP1. Due to the localization effect of Gal4, the distance between VP64 and the CMV promoter is reduced, thereby activating the expression of the downstream XPC and RUP2 genes. The presence of P2A allows for the independent translation of XPC and RUP2. To ensure that the XPC protein reaches a certain concentration before RUP2 inhibits the interaction between UVR8 and COP1, we have optimized the RUP2 sequence by replacing the codons in the RUP2 sequence with synonymous codons that are rare. By adjusting the number of replacements, the expression level of XPC is brought close to that found in normal somatic cells. Due to the repeated interaction and disengagement of UVR8 and COP1, XPC protein is stably maintained at an appropriate concentration. When the UV-B signal is removed, the UVR8 dimer cannot monomerize, ultimately achieving complete dissociation of RUP2 from UVR8 and COP1, constructing a UV-responsive XPC gene switch.
Fig6. Functional illustration of the P2A peptide. the DNA fragment encoding the P2A peptide is inserted into the middle of the coding region of two proteins, which can cause the peptide chain to undergo self-shearing after the completion of translation, splitting into two independently folded proteins
BBa K5182019—5×UAS-PhCMVmin-GFP-P2A-RUP2
In this composite part, the UAS sequence can be recognized by the Gal4 sequence, the GFP is used to detect the regulatory effect of the light control system, and the P2A sequence is used to promote the translation of the same mRNA strand to express GFP and RUP2, and the expressed RUP2 will play a negative feedback regulation on the system.This composite part has the same working principle as BBa_K5182020, BBa_K5182024.
Usage and Biology
Through the use of our optogenetic system and some of its components, we can regulate gene expression, significantly enhancing the targeting, safety, and efficiency of the gene therapy process. Our approach provides a novel perspective on the controllability and effectiveness of gene therapy for genetic diseases, particularly suitable for complex diseases requiring precise regulation of gene expression. In synthetic biology, integrating our optogenetic system with other synthetic biology modules enables the regulation of spatial and temporal gene expression through UV-B signals, facilitating the construction of more complex biological systems and providing new possibilities for the design of intricate biological circuits.
Fig7. Expanded applications of the UVR8-COP1-RUP2 system. (a). Mechanisms of action of photoregulatory systems; (b). Gene therapy based on optogenetic modulation; (c). Cell production factories based on optogenetic modulation)
Characterization
In terms of experimental design, we used a co-transfection group with the reporter gene plasmid, UVR8-VP64 plasmid, and COP1-NLS-Gal4 plasmid as the experimental group. We set up three control groups containing only the reporter gene plasmid, the reporter gene plasmid + UVR8-VP64 plasmid, and the reporter gene plasmid + COP1-NLS-Gal4 plasmid. This design was intended to rigorously demonstrate that the transcription activation system requires the coordinated function of all gene components, rather than the action of individual components. Additionally, we set up controls between irradiated and non-irradiated UV-B to illustrate the regulatory function of UV-B in the system. The schematic diagram of the transfection experimental design for the experimental and control groups is shown below:
Fig8. Schematic diagram of the transfection experimental design for the experimental group cell experiments
For this experiments, we observed GFP fluorescence under 488nm excitation and 560nm emission using an inverted fluorescence microscope. We found that only the experimental group co-transfected with all three plasmids and irradiated with UV-B showed significant fluorescence, while all other groups showed no significant fluorescence. Some control groups, such as the group with the target gene plasmid + UVR8-VP64 plasmid and irradiated with UV-B, showed a small amount of fluorescence, which we consider to be gene expression leakage within an acceptable range.
Fig9. GFP fluorescence observation image
Fig11. GFP fluorescent cell count per unit area
In this experiment, we verified the tunability of our optogenetic system, laying the foundation for subsequent experiments. Sequence and Features <pare 5rtinfo>BBa_K5182019 SequenceAndFeatures</partinfo>
References
- ↑ Gardner, L., & Deiters, A. (2012). Light-controlled synthetic gene circuits. Current opinion in chemical biology, 16(3-4), 292-299.
- ↑ Möglich, A., & Moffat, K. (2010). Engineered photoreceptors as novel optogenetic tools. Photochemical & photobiological sciences, 9(10), 1286-1300.
- ↑ Rizzini, L., Favory, J. J., Cloix, C., Faggionato, D., O’hara, A., Kaiserli, E., ... & Ulm, R. (2011). Perception of UV-B by the Arabidopsis UVR8 protein. Science, 332(6025), 103-106.
- ↑ Lin, R., & Wang, H. (2007). Targeting proteins for degradation by Arabidopsis COP1: teamwork is what matters. Journal of Integrative Plant Biology, 49(1), 35-42.
- ↑ Tilbrook, K., Arongaus, A. B., Binkert, M., Heijde, M., Yin, R., & Ulm, R. (2013). The UVR8 UV-B photoreceptor: perception, signaling and response. The Arabidopsis Book/American Society of Plant Biologists, 11.
- ↑ Struhl, K. (1995). Yeast transcriptional regulatory mechanisms. Annual review of genetics, 29(1), 651-674.
- ↑ Szymczak, A. L., & Vignali, D. A. (2005). Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert opinion on biological therapy, 5(5), 627-638.
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